The defect of biodefense mechanisms in bovine leukocyte adhesion deficiency syndrome (BLAD) as observed from signal transduction pathway

从信号转导途径观察牛白细胞粘附缺陷综合征（BLAD）生物防御机制的缺陷

• 批准号: 09460133
• 负责人: KUWABARA Mikinori
• 金额: $ 9.28万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09460133/
• 关键词: bovine leukocyte adhesion deficiency; BLAD; neutrophil; superoxide; signal transduction; NADPH oxidase; protein kinase; biodefense; 電子スピン共鳴法; 生体防御機構; ケミルミネッセンス; インテグリンファミリー; キナーゼ

This project was performed to clarify the signal transduction mechanisms for NADPH oxidase activation and phagocytotis by using normal neutrophils and those with bovine leukocyte adhesion deficiency (BLAD) which was genetic deficient in beta2-integrin CR3 corresponding to the receptor of complement iC3b. Various reagents to inhibit NADPH oxidase-related signal transduction were used for this purpose. We found that inhibitors of protein kinase C (PKC), phosphatidyl inositol 3-kinase (PI 3-kinase) and p38 mitogen-activated protein kinase (p38 MAPK) were dose-dependently inhibited superoxide generation from serum-opsonized zymosan (s-OZ)-stimulated neutrophils from BLAD and normal calves, although stimulation of BLAD neutrophils with s-OZ brought about the lower generation of superoxide than that of normal neutrophils with s-OZ.These results indicated that the lack of beta2-integrin CR3 did not influence signal transduction pathways of NADPH oxidase but reduced superoxide production of NA … More DPH oxidase. This reduced NADPH oxidase activity in BLAD was partially recovered by transfusion of CD 18-positive granulocytes to diseased calf. Furthennore, PI 3-kinase and p38 MAPK but not PKC were shown to be required for phagocytotic activity in normal neutrophils. Concerning the intracellular mechanisms of NADPH oxidase activation, the p47phox, one component of NADPH oxidase, is known to be extensively phosphorylated at serines that are located among its C-terminal region and the phosphorylation is a trigger for the activation of NADPH oxidase. Using site-directed mutagenesis of p47phox and p47phox-deficient B cells from human chronic granulomatous disease (CGD), it was shown that the phosphorylation of serines 303/304, 359/370 and possibly serine 379 must take place in order to activate the oxidase. These results seem to be important in not only understanding the signal transduction mechanism of NADPH oxidase activity but also development for therapy of BLAD and p47phox-deficient CGD by the granulocyte or gene transfusion. Less

通过使用正常嗜中性粒细胞和患有牛白细胞粘附性缺乏症（Blad）的那些在beta2-激素中缺乏与补充IC3B的接收者相对应的遗传性缺乏，以阐明NADPH氧化酶活性和吞噬的NADPH氧化酶激活和吞噬吞噬的信号转导机制。用于抑制NADPH氧化酶相关信号转导的各种试剂用于此目的。我们发现，蛋白激酶C（PKC），磷脂酰肌醇3-激酶（PI 3-激酶）和p38有丝分裂原活化蛋白激酶（p38 mapk）的抑制剂剂量依赖性地依赖性地抑制了超代性Zymosan（S-oz）的促进性超氧化物的超氧化物，并抑制了s-ozys-stim stim-stim staim staim-sprad andrars andrAd andrAd andrAd andrAd andrAd andrAd andrAd andrAd andrrAd andrrAdriLIl andRRADRILLILLILLIL，并抑制了BLAD的乳化剂，并抑制了阳离子的正常水平。与S-OZ的正常嗜中性粒细胞相比，具有S-oz的中性粒细胞带来的超氧化物具有低的生成氧化物。这些结果表明，缺乏beta2-融合蛋白CR3不影响NADPH氧化酶的信号转移途径，而是降低了Na…更多DPH氧化酶的超氧化物的产生。通过将CD 18阳性粒细胞输血向解散的小牛输血，部分回收了BLAD中的NADPH氧化酶活性。在正常嗜中性粒细胞中，吞噬活性是必需的。关于NADPH氧化酶激活的细胞内机制，已知p47phox是NADPH氧化酶的一个成分，在其C末端区域中被广泛地磷酸化，并且磷酸化是NADPH氧化酶激活的触发因素。使用人类慢性肉芽肿性疾病（CGD）的p47phox和p47phox缺陷型B细胞的位置定向诱变，证明丝氨酸303/304、359/370和可能的丝氨酸379的磷酸化必须发生以激活氧化酶。这些结果似乎不仅在理解NADPH氧化酶活动的信号传递机制方面也很重要，而且还开发了粒细胞或基因输血对骨的治疗和p47phox缺陷型CGD。较少的

项目成果

Johnson,J.L. et al.: "Activation of p47(PHOX), a cytosolic subunit of the leukocyte NADPH oxidase. Phosphorylation of ser-359 or ser-370 precedes phosphorylation at other sites and is required for activity." J Biol Chem. 273. 35147-35152 (1998)

约翰逊，J.L.

Osamu Inanami: "Oral administration of (-) catechin protects against ischemia-reperfusion-induced neuronal death in the gerbil. Free Radical Research" Free Radical Research. 29・4. 359-365 (1998)

Osamu Inanami：“口服（-）儿茶素可以防止沙鼠缺血再灌注引起的神经元死亡。自由基研究”29・4（1998）。

Inanami, O., Takahashi, K., Yoshito, A.and Kuwabara, M.: "H_2O_2-induced activation of SAPK/JNK regulated by phosphatidylinositol 3-kinase in Chinese hamster V79 cells." Antioxidant & Redox Signaling. (in press).

Inanami, O.、Takahashi, K.、Yoshito, A. 和 Kuwabara, M.：“中国仓鼠 V79 细胞中 H_2O_2 诱导的 SAPK/JNK 激活受磷脂酰肌醇 3-激酶调节。”

Kuwabara, M., Gouji, N.Inanami, O.Higuchi, H.and Nagahata, H.: An ESR study of superoxide generation in stimulated neutrophils from a calf with bovine leukocyte adhesion deficiency (BLAD).Modern Applications of EPR/ESR : From Biophysics to Material Scienc

Kuwabara，M.，Gouji，N.Inanami，O.Higuchi，H.和Nagahata，H.：牛白细胞粘附缺陷（BLAD）小牛受刺激中性粒细胞中超氧化物生成的ESR研究。EPR/ESR的现代应用

Hajime Nagahata: "Survival of transfused CD18-positive granulocytes and their chemiluminescent response in a heifer with leukocyte adhesion deficiency" Journal of Veterinary Medical Science. 60・2. 261-262 (1998)

Hajime Nagahata：“白细胞粘附缺陷的小母牛中输注的 CD18 阳性粒细胞的存活及其化学发光反应”《兽医医学杂志》60・2（1998 年）。